Gene delivery system and methods of use

ABSTRACT

A recombinant replication competent retrovirus for gene deliver and gene therapy is provided. The recombinant retrovirus has a heterologous nucleic acid sequence, a sequence encoding a cell- or tissue-specific ligand or a sequence for transcriptional targeting, or a combination of both a cell- or tissue-specific ligand and a cell- or tissue-specific transcriptional targeting sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuing application of Ser. No.09/409,650, filed Oct. 1, 1999, which claims priority from ProvisionalApplication Serial No. 60/102,933, filed Oct. 1, 1998, to whichapplication a priority claim is made under 35 U.S.C. §119(e). Therelated applications are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of viralvectors and specifically to a novel recombinant replication competentretrovirus useful for the transfer and expression of nucleic acidsequences in a targeted cell.

BACKGROUND OF THE INVENTION

[0003] The development of genetic vectors has heralded the fast-growingfield of somatic gene transfer. (Anderson, W. F., Science, 1984,226:401-409). Vectors based on simple retroviruses, such as the MoloneyLeukemia Virus (MoMLV), are often selected because they efficientlyintegrate into the genome of the target cell. Integration is thought tobe a prerequisite for long-term expression of the transduced gene.However, efficient gene transfer to tumor tissue has been a majorimpediment to treatment of cell proliferative disorders despite the useof viral vectors such as retroviruses.

[0004] In the early steps of infection, retroviruses deliver theirnucleoprotein core into the cytoplasm of the target cell. Here, reversetranscription of the viral genome takes place while the core maturesinto a preintegration complex. The complex must reach the nucleus toachieve integration of the viral DNA into the host cell chromosomes. Forsimple retroviruses (oncoretroviruses), this step requires thedissolution of the nuclear membrane at mitotic prophase, most likelybecause the bulky size of the preintegration complex prevents itspassive diffusion through the nuclear pores because there are no nuclearlocalization signals to facilitate active transport into the nucleus.

[0005] Currently retroviral vectors used for human gene therapy arereplication-defective and must be produced in packaging cells, whichcontain integrated wild type virus genome sequences and thus provide allof the structural elements necessary to assemble viruses (i.e., the gag,pol, and env gene products), but cannot encapsidate their own wild typevirus genomes due to a deletion of the packaging signal sequence (psi).Replication-defective virus vectors created by removal of the viralstructural genes and replacement with therapeutic genes are introducedinto the packaging cells; so long as these vectors contain the psisignal, they can take advantage of the structural proteins provided bythe cells and be encapsidated into virion. However, after infection of atarget cell, the vectors are incapable of secondary horizontalinfections of adjacent cells due to the deletion of the essential viralgenes.

[0006] The use of replication-defective vectors has been an importantsafeguard against the uncontrolled spread of virus, asreplication-competent retroviruses have been shown to cause malignanciesin primates (Donahue et al., J. Exp. Med., 1992, 176:1124-1135).However, replication-defective retroviral vectors are produced from thepackaging cells at titers on the order of only 10⁶⁻⁷ colony-formingunits (cfu) per ml, which is barely adequate for transduction in vivo.In fact, clinical trials for gene therapy of glioblastoma multiforme, ahighly malignant brain tumor, have encountered major problems inachieving adequate levels of tumor cell transduction, and despitepromising initial results in animal studies (Culver et al., Science,1992, 256:1550-1552). In order to increase transduction levels as muchas possible, instead of using a single shot of virus-containingsupernatant, the virus packaging cell line PA317 itself was injectedinto the brain tumors to constitutively produce retrovirus vectorscarrying the HSV-tk gene (Oldfield et al., Human Gene Therapy, 1993,4:39-69). Subsequently, the protocol was further modified to include adebulking procedure followed by multiple injection sites, as it wasfound that the virus vectors did not diffuse far enough from the site ofinitial injection. Despite these modifications, the transductionefficiency has been estimated to less than 1% of the tumor cell mass andany significant tumor destruction is presumed to be due to the potentbystander effect of the HSV-tk/ganciclovir treatment. Thus efficienttransduction of cancer cells in a solid tumor mass represents a majorproblem for cancer gene therapy.

[0007] Accordingly, there is a need for a gene transfer vector capableof high-level transduction in vivo, while limiting uncontrolled spreadof replication-competent virus which could result in insertionalmutagenesis and carcinogenesis.

SUMMARY OF THE INVENTION

[0008] The present invention provides recombinant replication competentretroviral vectors for gene delivery. The vectors provide a high-levelof transduction in vivo. The use of replication-competent vectors of theinvention allow efficient in vivo transduction. The incorporation ofcell-type targeting polynucleotide sequences into such vectors reduce oreliminate the native pathogenic potential of replication-competentretroviruses while improving their target cell specificity.

[0009] In one embodiment, the present invention provides a recombinantreplication competent retrovirus having a retroviral GAG protein; aretroviral POL protein; a retroviral ENV protein; a retroviral genomecomprising Long-Terminal Repeat (LTR) sequences at the 5′ and 3′ ends ofthe retroviral genome, wherein a target specific polynucleotide sequenceis contained within the LTR sequences at the 5′ and/or 3′ end of theretroviral genome, a heterologous nucleic acid sequence operably linkedto a regulatory nucleic acid sequence; and cis-acting nucleic acidsequences, and sequences encoding proteins, necessary for reversetranscription, packaging and integration in a target cell. The targetspecific polynucleotide sequence of the retroviral vector can be atissue-specific promoter sequence, for example a sequence associatedwith a growth regulatory gene, such as, for example, probasin. To targetthe retrovirus to a specific cell or tissue the retrovirus ENV proteincan further comprise a target-specific ligand sequence, which encodes,for example, an antibody, receptor, or ligand, such as, heregulin.

[0010] In another embodiment, the present invention provides arecombinant retroviral polynucleotide sequence, having a polynucleotidesequence encoding a GAG protein; a polynucleotide sequence encoding aPOL protein; a polynucleotide sequence encoding an ENV protein; apolynucleotide sequence comprising a Long Terminal Repeat (LTR) at the5′ and 3′ end of the retroviral polynucleotide sequence containing atarget specific polynucleotide sequence at the 5′ and or 3′ end; aheterologous polynucleotides sequence operably linked to a regulatorynucleic acid sequence; and cis acting polynucleotide sequence, as wellas sequences encoding proteins, necessary for reverse transcription,packaging and integration in a target cell. The target specificpolynucleotide sequence is a cell- or tissue-specific promoter sequencesuch as, for example, one associated with a growth regulatory gene orone associated with a cancer marker (e.g., probasin). The ENV sequencemay be further associated with a target-specific ligand polynucleotidesequence, for example a sequence encoding an antibody, a receptor (e.g.,a hormone receptor), or a ligand, such as, for example, heregulin.

[0011] In yet another embodiment, the present invention provides, amethod of treating a subject having a cell proliferative disorder, bycontacting the subject with a retrovirus, having a retroviral GAGprotein; a retroviral POL protein; a retroviral ENV protein; aretroviral genome comprising Long-Terminal Repeat (LTR) sequences at the5′ and 3′ end of the retroviral genome, wherein a target specificpolynucleotide sequence is contained within the LTR sequences at the 5′and/or 3′ end of the retroviral genome, a heterologous nucleic acidsequence operably linked to a regulatory nucleic acid sequence; andcis-acting nucleic acid sequences, as well as sequence encodingproteins, necessary for reverse transcription, packaging and integrationin a target cell. The target cell is preferably a cell having a cellproliferative disorder, such as a neoplastic cell.

[0012] These and other aspects of the present invention will be apparentto those of skill in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 (A) is a schematic illustration of the structure of wildtype (replication-competent) MoMLV retrovirus; LTR=long terminal repeat.(B) is a schematic representation of g1ZD-GFP and g1ZD-hygro, showingthe sizes of the IRES-transgene cassettes and the site of theirinsertion into the wild-type MLV genome. Arrows indicate location ofNheI sites used to digest DNA for Southern hybridization analysis, andwavy lines indicate regions of the vectors probed in hybridizationanalysis. Also shown is the sequences of g1ZD-GFP and g1ZD-hygro atlocations between env gene and IRES, IRES and GFP, and GFP and 3′ LTR.Bold letters indicate start or stop codons present within the junctions.

[0014]FIG. 2 is a schematic illustration of the structure of themodified MoMLV-based vectors of the present invention. A) A schematicdiagram of the structure of MoMLV-based replication-competent retroviral(RCR) vectors containing an internal ribosome entry site (IRES). B) Aschematic diagram of a targeted replication-competent retroviral vectors(RCRVs).

[0015]FIGS. 3A and 3B are graphs depicting a reverse transcriptase assayof MoMLV-based RCR vectors spread through NIH3T3 cells in culture.Mock=uninfected negative control; gIZAP=wild type replication-competentMoMLV virus; gIZD-gfp=replication-competent MoMLV virus with internalribosome entry site IRES at 3′ end of envelope gene and greenfluorescent protein (GFP) transgene; gIZD-puro=replication-competentMoMLV virus with IRES at 3′ end of envelope gene and puromycinresistance (PURO^(R)) transgene; gIZD-hygro=replication-competent MOMLVvirus with IRES at 3′ end of envelope gene and hygromycin resistance(HYGRO^(R)) transgene. FIG. 3B shows a comparison of Replication Kinetcsof Replication-competent Vectors and Wild Type Mo-MLV in Cultured Cells.

[0016]FIG. 4 are graphs showing fluorescence-activated cell sorter(FACS) analysis of GFP expression from gIZD-gfp RCR vector spread atvarious time points (Day 3, Day 5, and Day 8) after initial infection.

[0017]FIG. 5 is a graph depicting the stability of GFP transgeneexpression from the replication-competent gIZD-GFP vector over multipleserial passages.

[0018]FIG. 6 is a gel photo showing the stability of thereplication-competent gIZD-GFP vector over multiple serial passages.Lane P is a control sample containing either g1ZD-GFP (A) or g1ZD-hygro(B) plasmid DNA digested with NheI. The other lane numbers denote theserial passage number. (A) DNA from cells infected with g1ZD-GFP probedwith the GFP cDNA (left), and with the LTR-gag fragment (right). (B) DNAfrom cells infected with g1ZD-hygro probed with the hygromycinresistance gene (left), and with the LTR-gag fragment (right). Intact,full length gIZD-GFP is observed up to at least passage no. 11 (A, B).As shorter deletion mutant appears in passage nos/7-8 which then becomesthe dominant form. N denotes lanes containing Hirt DNA isolated fromuninfected NIH3T3 cells.

[0019]FIG. 7 is a schematic showing the experiment and resultsdemonstrating the highly efficient intra-tumoral spread of thereplication-competent gIZD-GFP vector within a subcutaneouslyestablished breast cancer model.

[0020]FIG. 8 is a schematic of a recombinant retroviral vector of theinvention containing a probasin promoter sequence.

[0021]FIGS. 9A and 9B show the construction of a recombinant replicationcompetent retrovirus of the invention targeted to prostate cancer cells.

[0022]FIG. 10 depicts a strategy used for determining prostatecell-specificity and androgen-inducibility of the probasin-LTR hybridpromoter.

[0023]FIG. 11 shows the results of the assay depicted in FIG. 10.

[0024]FIG. 12 depicts a strategy used for examining transcriptionalregulation of RCR vectors driven by the probasin-LTR hybrid promoter.

[0025]FIG. 13 shows the results of probasin-LTR drivenandrogen-responsive expression of the RCR vector GFP transgene afterinfection of TRAMP-C cells.

[0026]FIG. 14 shows the structure of RCR vectors with shorter IRESsequences.

[0027]FIG. 15 shows a comparison of GFP expression levels in cellsinfected with vectors utilizing three different IRES sequences.

[0028]FIG. 16 is a Southern Blot analysis of unintegrated proviral DNAfrom cultured cells serially infected with ZB-GFP (A) or ZV-GFP (B). Theprobes used were for the LTR-gag region of Mo-MLV.

[0029]FIG. 17 is a schematic of Z-domain targeted RCR vectors. Bothvectors contain 2 tandem copies of the Z domain of protein A within thePRR of the envelope gene. In ZE-GFP, GFP translation is driven by theEMCV IRES, and in ZV-A-GFP by the VEGF IRES.

[0030]FIG. 18 is a schematic showing the envelope structure of RCRvectors containing anti-HER2 scFv. ss: signal sequence; SU: surfaceprotein; RBD: receptor-binding domain; PRR: proline-rich region; C-SU:C-terminus of SU; TM: transmembrane protein; spacer: a synthetic 6 aminoacid spacer.

DETAILED DESCRIPTION OF THE INVENTION

[0031] To facilitate understanding of the invention, a number of termsare defined below.

[0032] “Polynucleotide” or “nucleic acid sequence” refers to a polymericform of nucleotides at least 9 bases in length. By “isolated nucleicacid sequence” is meant a polynucleotide that is not immediatelycontiguous with either of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived. Theterm therefore includes, for example, a recombinant DNA or RNA which isincorporated into a viral vector. The nucleotides of the invention canbe ribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double stranded forms of DNA.

[0033] The term polynucleotide(s) generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas used herein refers to, among others, single-and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions.

[0034] In addition, polynucleotide as used herein can also refer totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

[0035] As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

[0036] It will be appreciated that a great variety of modifications havebeen made to DNA and RNA that serve many useful purposes known to thoseof skill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

[0037] The present invention provides a recombinantreplication-competent retrovirus capable of infecting targeted cells.The virus is useful for the in vivo and ex vivo transfer and expressionof genes and nucleic acid sequences (e.g., in dividing and non-dividingcells). In particular, the present retroviral vectors are useful intargeting specific cell types including, but not limited to, neoplasticcells or cells having cell-proliferative disorders.

[0038] The present invention has many utilities. For example, theretrovirus and methods of the present invention can be used to provide atherapeutic product to a subject, for providing gene delivery of anon-therapeutic protein or a therapeutic protein to a subject, as wellas in in vitro studies to provide a cell with a gene for expression of agene product. Such in vitro methods are useful, for example, in proteinproduction and the study of regulation and interaction of cis-actingproducts, and polypeptides.

[0039] Retroviruses

[0040] Retroviruses are RNA viruses wherein the viral genome is RNA.When a host cell is infected with a retrovirus, the genomic RNA isreverse transcribed into a DNA intermediate which is integrated veryefficiently into the chromosomal DNA of infected cells. The integratedDNA intermediate is referred to as a provirus. The family Retroviridaeare enveloped single-stranded RNA viruses that typically infect mammals,such as, for example, bovines, monkeys, sheep, and humans, as well asavian species. Retroviruses are unique among RNA viruses in that theirmultiplication involves the synthesis of a DNA copy of the RNA which isthen integrated into the genome of the infected cell.

[0041] The Retroviridae family consists of three groups: thespumaviruses (or foamy viruses) such as the human foamy virus (HFV); thelentiviruses, as well as visna virus of sheep; and the oncoviruses(although not all viruses within this group are oncogenic). The term“lentivirus” is used in its conventional sense to describe a genus ofviruses containing reverse transcriptase. The lentiviruses include the“immunodeficiency viruses” which include human immunodeficiency virus(HIV) type 1 and type 2 (HIV-1 and HIV-2) and simian immunodeficiencyvirus (SIV). The oncoviruses are further subdivided into groups A, B, Cand D on the basis of particle morphology, as seen under the electronmicroscope during viral maturation. A-type particles represent theimmature particles of the B- and D-type viruses seen in the cytoplasm ofinfected cells. These particles are not infectious. B-type particles budas mature virion from the plasma membrane by the enveloping ofintracytoplasmic A-type particles. At the membrane they possess atoroidal core of ˜75 nm, from which long glycoprotein spikes project.After budding, B-type particles contain an eccentrically located,electron-dense core. The prototype B-type virus is mouse mammary tumorvirus (MMTV). No intracytoplasmic particles can be observed in cellsinfected by C-type viruses. Instead, mature particles bud directly fromthe cell surface via a crescent ‘C’-shaped condensation which thencloses on itself and is enclosed by the plasma membrane. Envelopeglycoprotein spikes may be visible, along with a uniformlyelectron-dense core. Budding may occur from the surface plasma membraneor directly into intracellular vacuoles. The C-type viruses are the mostcommonly studied and include many of the avian and murine leukemiaviruses (MLV). Bovine leukemia virus (BLV), and the human T-cellleukemia viruses types I and II (HTLV-I/II) are similarly classified asC-type particles because of the morphology of their budding from thecell surface. However, they also have a regular hexagonal morphology andmore complex genome structures than the prototypic C-type viruses suchas the murine leukemia viruses (MLV). D-type particles resemble B-typeparticles in that they show as ring-like structures in the infected cellcytoplasm, which bud from the cell surface, but the virion incorporateshort surface glycoprotein spikes. The electron-dense cores are alsoeccentrically located within the particles. Mason Pfizer monkey virus(MPMV) is the prototype D-type virus.

[0042] Retroviruses are defined by the way in which they replicate theirgenetic material. During replication the RNA is converted into DNA.Following infection of the cell a double-stranded molecule of DNA isgenerated from the two molecules of RNA which are carried in the viralparticle by the molecular process known as reverse transcription. TheDNA form becomes covalently integrated in the host cell genome as aprovirus, from which viral RNAs are expressed with the aid of cellularand/or viral factors. The expressed viral RNAs are packaged intoparticles and released as infectious virion.

[0043] The retrovirus particle is composed of two identical RNAmolecules. Each wild-type genome has a positive sense, single-strandedRNA molecule, which is capped at the 5′ end and polyadenylated at the 3′tail. The diploid virus particle contains the two RNA strands complexedwith gag proteins, viral enzymes (pol gene products) and host tRNAmolecules within a ‘core’ structure of gag proteins. Surrounding andprotecting this capsid is a lipid bilayer, derived from host cellmembranes and containing viral envelope (env) proteins. The env proteinsbind to a cellular receptor for the virus and the particle typicallyenters the host cell via receptor-mediated endocytosis and/or membranefusion.

[0044] After the outer envelope is shed, the viral RNA is copied intoDNA by reverse transcription. This is catalyzed by the reversetranscriptase enzyme encoded by the pol region and uses the host celltRNA packaged into the virion as a primer for DNA synthesis. In this waythe RNA genome is converted into the more complex DNA genome.

[0045] The double-stranded linear DNA produced by reverse transcriptionmay, or may not, have to be circularized in the nucleus. The provirusnow has two identical repeats at either end, known as the long terminalrepeats (LTR). The termini of the two LTR sequences produces the siterecognized by a pol product—the integrase protein—which catalyzesintegration, such that the provirus is always joined to host DNA twobase pairs (bp) from the ends of the LTRS. A duplication of cellularsequences is seen at the ends of both LTRs, reminiscent of theintegration pattern of transposable genetic elements. Integration isthought to occur essentially at random within the target cell genome.However, by modifying the long-terminal repeats it is possible tocontrol the integration of a retroviral genome.

[0046] Transcription, RNA splicing and translation of the integratedviral DNA is mediated by host cell proteins. Variously splicedtranscripts are generated. In the case of the human retroviruses HIV-1/2and HTLV-I/II viral proteins are also used to regulate gene expression.The interplay between cellular and viral factors is important in thecontrol of virus latency and the temporal sequence in which viral genesare expressed.

[0047] Retroviruses can be transmitted horizontally and vertically.Efficient infectious transmission of retroviruses requires theexpression on the target cell of receptors which specifically recognizethe viral envelope proteins, although viruses may usereceptor-independent, nonspecific routes of entry at low efficiency. Inaddition, the target cell type must be able to support all stages of thereplication cycle after virus has bound and penetrated. Verticaltransmission occurs when the viral genome becomes integrated in the germline of the host. The provirus will then be passed from generation togeneration as though it were a cellular gene. Hence endogenousproviruses become established which frequently lie latent, but which canbecome activated when the host is exposed to appropriate agents.

[0048] Replication Competent Recombinant Retroviruses

[0049] As mentioned above, the integrated DNA intermediate is referredto as a provirus. Prior gene therapy or gene delivery systems usemethods and retroviruses that require transcription of the provirus andassembly into infectious virus while in the presence of an appropriatehelper virus or in a cell line containing appropriate sequences enablingencapsidation without coincident production of a contaminating helpervirus. As described below, a helper virus is not required for theproduction of the recombinant retrovirus of the present invention, sincethe sequences for encapsidation are provided in the genome thusproviding a replication competent retroviral vector for gene delivery ortherapy.

[0050] The retroviral genome and the proviral DNA of the presentinvention have at least three genes: the gag, the pol, and the env,which are flanked by two long terminal repeat (LTR) sequences containingcis-acting sequences such as psi. The gag gene encodes the internalstructural (matrix, capsid, and nucleocapsid) proteins; the pol geneencodes the RNA-directed DNA polymerase (reverse transcriptase),protease and integrase; and the env gene encodes viral envelopeglycoproteins. The 5′ and 3′ LTRs serve to promote transcription andpolyadenylation of the virion RNAs. The LTR contains all othercis-acting sequences necessary for viral replication. Lentiviruses haveadditional genes including vif, vpr, tat, rev, vpu, nef, and vpx (inHIV-1, HIV-2 and/or SIV).

[0051] Adjacent to the 5′ LTR are sequences necessary for reversetranscription of the genome (the tRNA primer binding site) and forefficient encapsidation of viral RNA into particles (the Psi site). Ifthe sequences necessary for encapsidation (or packaging of retroviralRNA into infectious virion) are missing from the viral genome, theresult is a cis defect which prevents encapsidation of genomic viralRNA. This type of modified vector is what has typically been used inprior gene delivery systems (i.e., systems lacking elements which arerequired for encapsidation of the virion).

[0052] In a first embodiment, the invention provides a recombinantretrovirus capable of infecting a non-dividing cell, a dividing cell, ora cell having a cell proliferative disorder. The recombinant replicationcompetent retrovirus of the present invention comprises a polynucleotidesequence having a viral GAG, a viral POL, a viral ENV, a heterologouspolynucleotide and one or more targeting polynucleotide sequence forcell- or tissue-specific targeting of the retrovirus to a particulartissue, cell or cell type, as described herein.

[0053] The heterologous nucleic acid sequence is operably linked to aregulatory nucleic acid sequence. As used herein, the term“heterologous” nucleic acid sequence or transgene refers to a sequencethat does not normally exist in the wild (e.g., in the wild-typeretrovirus) or a sequence that originates from a foreign species, or, iffrom the same species, it may be substantially modified from itsoriginal form. Alternatively, an unchanged nucleic acid sequence that isnot normally expressed in a cell is a heterologous nucleic acidsequence.

[0054] Depending upon the intended use of the retroviral vector of thepresent invention any number of heterologous polynucleotide or nucleicacid sequences may be inserted into the retroviral vector. For example,for in vitro studies commonly used marker genes or reporter genes may beused, including, antibiotic resistance and fluorescent molecules (e.g.,GFP). Additional polynucleotide sequences encoding any desiredpolypeptide sequence may also be inserted into the vector of the presentinvention. Where in vivo delivery of a heterologous nucleic acidsequence is sought both therapeutic and non-therapeutic sequences may beused. For example, the heterologous sequence can encode a therapeuticmolecule including antisense molecules or ribozymes directed to aparticular gene associated with a cell proliferative disorder, theheterologous sequence can be a suicide gene (e.g., HSV-tk or PNP), or atherapeutic protein (e.g., Factor IX). Other therapeutic proteinsapplicable to the present invention are easily identified in the art(see for example, R. Crystal, Science 270:404-410 (1995)).

[0055] Thus, the recombinant virus of the invention is capable oftransferring a nucleic acid sequence into a target cell. The termnucleic acid sequence refers to any nucleic acid molecule, includingDNA, RNA or modified nucleic acid sequences. The nucleic acid moleculemay be derived from a variety of sources, including DNA, cDNA, syntheticDNA, RNA, or combinations thereof. Such nucleic acid sequences maycomprise genomic DNA which may or may not include naturally occurringintrons. Moreover, such genomic DNA may be obtained in association withpromoter regions, introns, or poly A sequences. Genomic DNA may beextracted and purified from suitable cells by means well known in theart. Alternatively, messenger RNA (mRNA) can be isolated from cells andused to produce cDNA by reverse transcription or other means.

[0056] The term “regulatory nucleic acid sequence” refers collectivelyto promoter sequences, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, origins ofreplication, internal ribosome entry sites (“IRES”), enhancers, and thelike, which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesecontrol sequences need always be present so long as the selected codingsequence is capable of being replicated, transcribed and translated inan appropriate host cell. One skilled in the art can readily identifyregulatory nucleic acid sequence from public databases and materials.Furthermore, one skilled in the art can identify a regulatory sequencethat is applicable for the intended use, for example, in vivo, ex vivo,or in vitro.

[0057] The term “promoter region” is used herein in its ordinary senseto refer to a nucleotide region comprising a DNA regulatory sequence,wherein the regulatory sequence is derived from a gene which is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. The regulatory sequence may behomologous or heterologous to the desired gene sequence. For example, awide range of promoters may be utilized, including viral or mammalianpromoter. Preferably the regulatory sequences is an IRES sequence.

[0058] The term “operably linked” refers to functional linkage betweenthe regulatory sequence and the heterologous nucleic acid sequence. Theheterologous sequence can be linked to a promoter, resulting in achimeric gene. The heterologous nucleic acid sequence is preferablyunder control of either the viral LTR promoter-enhancer signals or of aninternal promoter, and retained signals within the retroviral LTR canstill bring about efficient integration of the vector into the host cellgenome. Accordingly, the recombinant retroviral vectors of theinvention, the desired sequences, genes and/or gene fragments can beinserted at several sites and under different regulatory sequences. Forexample, a site for insertion can be the viral enhancer/promoterproximal site (i.e., 5′ LTR-driven gene locus). Alternatively, thedesired sequences can be inserted into a regulatory sequence distal site(e.g., the IRES sequence 3′ to the env gene). Other distal sites includeviral promoter sequences, where the expression of the desired sequenceor sequences is through splicing of the promoter proximal cistron, aninternal heterologous promoter as SV40 or CMV, or an internal ribosomeentry site (IRES).

[0059] In one embodiment, the retroviral genome of the present inventioncontains an IRES comprising a cloning site for insertion of a desiredpolynucleotide sequence, preferably the IRES is 3′ to the env gene inthe retroviral vector. Accordingly, a heterologous polynucleotidesequence encoding a desired polypeptide may be operably linked to theIRES. An example of polynucleotide sequence which may be operably linkedto the IRES include green fluorescent protein (GFP) or a selectablemarker gene. Marker genes are utilized to assay for the presence of thevector, and thus, to confirm infection and integration. Typicalselection genes encode proteins that confer resistance to antibioticsand other toxic substances, e.g., histidinol, puromycin, hygromycin,neomycin, methotrexate, and other reporter genes known in the art. Otherpolynucleotide sequence which may be linked to the IRES include, forexample, suicide genes, such as PNP and HSV-thymidine kinase (FIG. 2),polynucleotide sequences that encode an antisense molecule, orpolynucleotides sequences that encode a ribosome.

[0060] It can be advantageous to have at one's disposal more efficaciousgene therapy vectors capable, in particular, of producing severalproteins of interest efficiently. However, the presence of severalpromoters within the same vector very often manifests itself in areduction or even a loss of expression over time. This is due to awell-known phenomenon of interference between promoter sequences. Inthis context, the publication of International Application WO93/03143proposes a solution to this problem which consists in employing an IRES.It describes a dicistonic retroviral vector for the expression of twogenes of interest placed under the control of the same promoter. Forexample, the presence of a picornavirus IRES site between these genespermits the production of the expression product originating from thesecond gene of interest by internal initiation of the translation of thedicistronic mRNA (see Morgan et al., Nucleic Acids Research, 20:(6)1293-1299 (1992)).

[0061] Normally, the entry of ribosomes into messenger RNA takes placevia the cap located at the 5′ end of all eukaryotic mRNAs. However,there are exceptions to this universal rule. The absence of a cap insome viral mRNAs suggests the existence of alternative structurespermitting the entry of ribosomes at an internal site of these RNAs. Todate, a number of these structures, designated IRES on account of theirfunction, have been identified in the 5′ noncoding region of uncappedviral mRNAs, such as that, in particular, of picornaviruses such as thepoliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol., 8,1103-1112) and the EMCV virus (encephalo-myocarditis virus (Jang et al.,J. Virol., 1988, 62, 2636-2643). The present invention provides the useof an IRES in the context of a replication-competent retroviral vector.

[0062] In another embodiment a targeting polynucleotide sequence isincluded as part of the recombinant retroviral vector of the presentinvention. The targeting polynucleotide sequence is a targeting ligand(e.g., peptide hormones such as heregulin, a single-chain antibodies, areceptor or a ligand for a receptor), a tissue-specific or cell-typespecific regulatory element (e.g., a tissue-specific or cell-typespecific promoter or enhancer), or a combination of a targeting ligandand a tissue-specific/cell-type specific regulatory element. Preferably,the targeting ligand is operably linked to the env protein of theretrovirus, creating a chimeric retroviral env protein. The viral GAG,viral POL and viral ENV proteins can be derived from any suitableretrovirus (e.g.,MLV or lentivirus-derived). In another embodiment, theviral ENV protein is non-retrovirus-derived (e.g., CMV or VSV).

[0063] The recombinant retrovirus of the invention is thereforegenetically modified in such a way that the virus is targeted to aparticular cell type (e.g., smooth muscle cells, hepatic cells, renalcells, fibroblasts, keratinocytes, mesenchymal stem cells, bone marrowcells, chondrocyte, epithelial cells, intestinal cells, neoplastic cellsand others known in the art) such that the nucleic acid genome isdelivered to a target non-dividing, a target dividing cell, or a targetcell having a cell proliferative disorder. Targeting can be achieved intwo ways. The first way directs the retrovirus to a target cell bypreferentially binding to cells having a molecule on the externalsurface of the cell. This method of targeting the retrovirus utilizesexpression of a targeting ligand on the coat of the retrovirus to assistin targeting the virus to cells or tissues that have a receptor orbinding molecule which interacts with the targeting ligand on thesurface of the retrovirus. After infection of a cell by the virus, thevirus injects its nucleic acid into the cell and the retrovirus geneticmaterial can integrate into the host cell genome. The second method fortargeting uses cell- or tissue-specific regulatory elements topreferentially promote expression and transcription of the viral genomein a targeted cell which actively utilizes the regulatory elements, asdescribed more fully below. The transferred retrovirus genetic materialis then transcribed and translated into proteins within the host cell.The targeting regulatory element is preferably linked to the 5′ and/or3′ LTR, creating a chimeric LTR.

[0064] By inserting a heterologous nucleic acid sequence of interestinto the viral vector of the invention, along with another gene whichencodes, for example, the ligand for a receptor on a specific targetcell, the vector is now target specific. Viral vectors can be madetarget specific by attaching, for example, a sugar, a glycolipid, or aprotein. Targeting can be accomplished by using an antibody to targetthe viral vector. Those of skill in the art will know of, or can readilyascertain, specific polynucleotide sequences which can be inserted intothe viral genome or proteins which can be attached to a viral envelopeto allow target specific delivery of the viral vector containing thenucleic acid sequence of interest.

[0065] Thus, the present invention, includes in one embodiment, achimeric env protein comprising a retroviral env protein operably linkedto a targeting polypeptide. The targeting polypeptide can be a cellspecific receptor molecule, a ligand for a cell specific receptor, anantibody or antibody fragment to a cell specific antigenic epitope orany other ligand easily identified in the art which is capable ofbinding or interacting with a target cell. Examples of targetingpolypeptides or molecules include bivalent antibodies usingbiotin-streptavidin as linkers (Etienne-Julan et al., J. Of GeneralVirol., 73, 3251-3255 (1992); Roux et al., Proc. Natl. Acad. Sci USA 86,9079-9083 (1989)), recombinant virus containing in its envelope asequence encoding a single-chain antibody variable region against ahapten (Russell et al., Nucleic Acids Research, 21, 1081-1085 (1993)),cloning of peptide hormone ligands into the retrovirus envelope(Kasahara et al., Science, 266, 1373-1376 (1994)), chimeric EPO/envconstructs (Kasahara et al., 1994), single-chain antibody against thelow density lipoprotein (LDL) receptor in the ecotropic MLV envelope,resulting in specific infection of HeLa cells expressing LDL receptor(Somia et al., Proc. Natl. Acad. Sci USA, 92, 7570-7574 (1995)),similarly the host range of ALV can be altered by incorporation of anintegrin ligand, enabling the virus to now cross species to specificallyinfect rat glioblastoma cells (Valsesia-Wittmann et al., J. Virol. 68,4609-4619 (1994)), and Dornberg and co-workers (Chu and Dornburg, J.Virol 69, 2659-2663 (1995)) have reported tissue-specific targeting ofspleen necrosis virus (SNV), an avian retrovirus, using envelopescontaining single-chain antibodies directed against tumor markers.

[0066] The invention provides a method of producing a recombinantretrovirus capable of infecting a target cell comprising transfecting asuitable host cell with the following: a vector comprising apolynucleotide sequence encoding a viral gag, a viral pol and a viralenv, wherein the vector contains a cloning site for introduction of aheterologous gene, operably linked to a regulatory nucleic acidsequence, and recovering the recombinant virus. An illustration of theindividual vectors used in the method of the invention is shown in FIGS.1 and 2.

[0067] The retrovirus and methods of the invention provide a replicationcompetent retrovirus that does not require helper virus or additionalnucleic acid sequence or proteins in order to propagate and producevirion. For example, the nucleic acid sequences of the retrovirus of thepresent invention encode, for example, a group specific antigen andreverse transcriptase, (and integrase and protease-enzymes necessary formaturation and reverse transcription), respectively, as discussed above.The viral gag and pol can be derived from a lentivirus, such as HIV oran oncovirus such as MoMLV. In addition, the nucleic acid genome of theretrovirus of the present invention includes a sequence encoding a viralenvelope (ENV) protein. The env gene can be derived from anyretroviruses. The env may be an amphotropic envelope protein whichallows transduction of cells of human and other species, or may be anecotropic envelope protein, which is able to transduce only mouse andrat cells. Further, it may be desirable to target the recombinant virusby linkage of the envelope protein with an antibody or a particularligand for targeting to a receptor of a particular cell-type. Asmentioned above, retroviral vectors can be made target specific byinserting, for example, a glycolipid, or a protein. Targeting is oftenaccomplished by using an antibody to target the retroviral vector to anantigen on a particular cell-type (e.g., a cell type found in a certaintissue, or a cancer cell type). Those of skill in the art will know of,or can readily ascertain without undue experimentation, specific methodsto achieve delivery of a retroviral vector to a specific target. In oneembodiment, the env gene is derived from a non-retrovirus (e.g., CMV orVSV). Examples of retroviral-derived env genes include, but are notlimited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon apeleukemia virus (GaLV), human immunodeficiency virus (HIV) and RousSarcoma Virus (RSV). Other env genes such as Vesicular stomatitis virus(VSV) (Protein G), cytomegalovirus envelope (CMV), or influenza virushemagglutinin (HA) can also be used.

[0068] Unlike recombinant retroviruses produced by standard methods inthe art that are defective and require assistance in order to produceinfectious vector particles, the present invention provides a retrovirusthat is replication-competent.

[0069] In another embodiment, the present invention provides retroviralvectors that are targeted using regulatory sequences. Cell- ortissue-specific regulatory sequences (e.g., promoters) can be utilizedto target expression of gene sequences in specific cell populations.Suitable mammalian and viral promoters for the present invention areavailable in the art. Accordingly, in one embodiment, the presentinvention provides a retrovirus having tissue-specific promoter elementsat the 5′ and 3′ end of the retroviral genome. Preferably, thetissue-specific regulatory elements/sequences are in the U3 region ofthe LTR of the retroviral genome, including for example cell- ortissue-specific promoters and enhancers to neoplastic cells (e.g., tumorcell-specific enhancers and promoters), and inducible promoters (e.g.,tetracycline). Transcription control sequences of the present inventioncan also include naturally occurring transcription control sequencesnaturally associated with a gene encoding a superantigen, a cytokine ora chemokine of the present invention.

[0070] “Tissue-specific regulatory elements” are regulatory elements(e.g., promoters) that are capable of driving transcription of a gene inone tissue while remaining largely “silent” in other tissue types. Itwill be understood, however, that tissue-specific promoters may have adetectable amount of “background” or “base” activity in those tissueswhere they are silent. The degree to which a promoter is selectivelyactivated in a target tissue can be expressed as a selectivity ratio(activity in a target tissue/activity in a control tissue). In thisregard, a tissue specific promoter useful in the practice of the presentinvention typically has a selectivity ratio of greater than about 5.Preferably, the selectivity ratio is greater than about 15.

[0071] It will be further understood that certain promoters, while notrestricted in activity to a single tissue type, may nevertheless showselectivity in that they may be active in one group of tissues, and lessactive or silent in another group. Such promoters are also termed“tissue specific”, and are contemplated for use with the presentinvention. For example, promoters that are active in a variety ofcentral nervous system (CNS) neurons may be therapeutically useful inprotecting against damage due to stroke, which may effect any of anumber of different regions of the brain. Accordingly, thetissue-specific regulatory elements used in the present invention, haveapplicability to regulation of the heterologous proteins as well as aapplicability as a targeting polynucleotide sequence in the presentretroviral vectors.

[0072] Tissue-specific promoters may be derived, for example, frompromoter regions of genes that are differentially expressed in differenttissues. For example, a variety of promoters have been identified whichare suitable for up regulating expression in cardiac tissue. Included,for example, are the cardiac α-myosin heavy chain (AMHC) promoter andthe cardiac α-actin promoter. Other examples of tissue-specificregulatory elements include, tissue-specific promoters, such asmilk-specific (whey), pancreatic (insulin or elastase), actin promoterin smooth muscle cells or neuronal (myelin basic protein) promoters.Through the use of promoters, such as milk-specific promoters,recombinant retroviruses may be isolated directly from the biologicalfluid of the progeny.

[0073] In addition, numerous gene therapy methods, that take advantageof retroviral vectors, for treating a wide variety of diseases arewell-known in the art (see, e.g., U.S. Pat. Nos. 4,405,712 and4,650,764; Friedmann, 1989, Science, 244:1275-1281; Mulligan, 1993,Science, 260:926-932, R. Crystal, 1995, Science 270:404-410, each ofwhich are incorporated herein by reference in their entirety). Anincreasing number of these methods are currently being applied in humanclinical trials (Morgan, R., 1993, BioPharm, 6(1) :32-35; see also TheDevelopment of Human Gene Therapy, Theodore Friedmann, Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. ISBN0-87969-528-5, which is incorporated herein by reference in itsentirety). The safety of these currently available gene therapyprotocols can be substantially increased by using retroviral vectors ofthe present invention. For example, where the retroviral vector infectsa non-targeted cell, the retroviral genome will integrate but will notbe transcribed. However, when the retroviral vector containing a tissuespecific regulatory element infects a targeted cell the active tissuespecific promoter will result in transcription and translation of theviral genome.

[0074] The phrase “non-dividing” cell refers to a cell that does not gothrough mitosis. Non-dividing cells may be blocked at any point in thecell cycle, (e.g., G₀/G₁, G₁/S, G₂/M), as long as the cell is notactively dividing. For ex vivo infection, a dividing cell can be treatedto block cell division by standard techniques used by those of skill inthe art, including, irradiation, aphidocolin treatment, serumstarvation, and contact inhibition. However, it should be understoodthat ex vivo infection is often performed without blocking the cellssince many cells are already arrested (e.g., stem cells). For example, arecombinant lentivirus vector of the invention is capable of infectingany non-dividing cell, regardless of the mechanism used to block celldivision or the point in the cell cycle at which the cell is blocked.Examples of pre-existing non-dividing cells in the body includeneuronal, muscle, liver, skin, heart, lung, and bone marrow cells, andtheir derivatives. For dividing cells onco-retroviral vectors can beused.

[0075] By “dividing” cell is meant a cell that undergoes active mitosis,or meiosis. Such dividing cells include stem cells, skin cells (e.g.,fibroblasts and keratinocytes), gametes, and other dividing cells knownin the art. Of particular interest and encompassed by the term dividingcell are cells having cell proliferative disorders, such as neoplasticcells. The term “cell proliferative disorder” refers to a conditioncharacterized by an abnormal number of cells. The condition can includeboth hypertrophic (the continual multiplication of cells resulting in anovergrowth of a cell population within a tissue) and hypotrophic (a lackor deficiency of cells within a tissue) cell growth or an excessiveinflux or migration of cells into an area of a body. The cellpopulations are not necessarily transformed, tumorigenic or malignantcells, but can include normal cells as well. Cell proliferativedisorders include disorders associated with an overgrowth of connectivetissues, such as various fibrotic conditions, including scleroderma,arthritis and liver cirrhosis. Cell proliferative disorders includeneoplastic disorders such as head and neck carcinomas. Head and neckcarcinomas would include, for example, carcinoma of the mouth,esophagus, throat, larynx, thyroid gland, tongue, lips, salivary glands,nose, paranasal sinuses, nasopharynx, superior nasal vault and sinustumors, esthesioneuroblastoma, squamous call cancer, malignant melanoma,sinonasal undifferentiated carcinoma (SNUC) or blood neoplasia. Alsoincluded are carcinoma's of the regional lymph nodes including cervicallymph nodes, prelaryngeal lymph nodes, pulmonary juxtaesophageal lymphnodes and submandibular lymph nodes (Harrison's Principles of InternalMedicine (eds., Isselbacher, et al., McGraw-Hill, Inc., 13th Edition,pp1850-1853, 1994). Other cancer types, include, but are not limited to,lung cancer, colon-rectum cancer, breast cancer, prostate cancer,urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreaticcancer, leukemia, melanoma, stomach cancer and ovarian cancer.

[0076] The present invention also provides gene therapy for thetreatment of cell proliferative disorders. Such therapy would achieveits therapeutic effect by introduction of an appropriate therapeuticpolynucleotide sequence (e.g., antisense, ribozymes, suicide genes),into cells of subject having the proliferative disorder. Delivery ofpolynucleotide constructs can be achieved using the recombinantretroviral vector of the present invention, particularly if ti si basedon MLV, which will is capable of infecting dividing cells.

[0077] In addition, the therapeutic methods (e.g., the gene therapy orgene delivery methods) as described herein can be performed in vivo orex vivo. It may be preferable to remove the majority of a tumor prior togene therapy, for example surgically or by radiation.

[0078] Thus, the invention provides a recombinant retrovirus capable ofinfecting a non-dividing cell, a dividing cell or a neoplastic cellcomprising a viral GAG; a viral POL; a viral ENV; a heterologous nucleicacid sequence operably linked to a regulatory nucleic acid sequence; andcis-acting nucleic acid sequences necessary for packaging, reversetranscription and integration. The recombinant retrovirus can be alentivirus, such as HIV, or can be an oncovirus. As described above forthe method of producing a recombinant retrovirus, the recombinantretrovirus of the invention may further include at least one of VPR,VIF, NEF, VPX, TAT, REV, and VPU protein. While not wanting to be boundby a particular theory, it is believed that one or more of thesegenes/protein products are important for increasing the viral titer ofthe recombinant retrovirus produced (e.g., NEF) or may be necessary forinfection and packaging of virion, depending on the packaging cell linechosen (e.g., VIF).

[0079] The invention also provides a method of nucleic acid transfer toa target cell to provide expression of a particular nucleic acidsequence (e.g., a heterologous sequence). Therefore, in anotherembodiment, the invention provides a method for introduction andexpression of a heterologous nucleic acid sequence in a target cellcomprising infecting the target cell with the recombinant virus of theinvention and expressing the heterologous nucleic acid sequence in thetarget cell. As mentioned above, the target cell can be any cell typeincluding dividing, non-dividing, neoplastic, immortalized, modified andother cell types recognized by those of skill in the art, so long asthey are capable of infection by a retrovirus.

[0080] It may be desirable to modulate the expression of a gene in acell by the introduction of a nucleic acid sequence (e.g., theheterologous nucleic acid sequence) by the method of the invention,wherein the nucleic acid sequence give rise, for example, to anantisense or ribozyme molecule. The term “modulate” envisions thesuppression of expression of a gene when it is over-expressed, oraugmentation of expression when it is under-expressed. Where a cellproliferative disorder is associated with the expression of a gene,nucleic acid sequences that interfere with the gene's expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid, ribozymes, or triplex agents to blocktranscription or translation of a specific mRNA, either by masking thatmRNA with an antisense nucleic acid or triplex agent, or by cleaving itwith a ribozyme.

[0081] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetcell. The use of antisense methods to inhibit the in vitro translationof genes is well known in the art (Marcus-Sakura, Anal.Biochem.,172:289, 1988).

[0082] The antisense nucleic acid can be used to block expression of amutant protein or a dominantly active gene product, such as amyloidprecursor protein that accumulates in Alzheimer's disease. Such methodsare also useful for the treatment of Huntington's disease, hereditaryParkinsonism, and other diseases. Of particular interest are theblocking of genes associated with cell-proliferative disorders.Antisense nucleic acids are also useful for the inhibition of expressionof proteins associated with toxicity.

[0083] Use of an oligonucleotide to stall transcription is known as thetriplex strategy since the oligomer winds around double-helical DNA,forming a three-strand helix. Therefore, these triplex compounds can bedesigned to recognize a unique site on a chosen gene (Maher, et al.,Antisense Res. and Dev., 1(3) :227, 1991; Helene, C., Anticancer DrugDesign, 6(6):569, 1991).

[0084] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0085] It may be desirable to transfer a nucleic acid encoding abiological response modifier. Included in this category areimmunopotentiating agents including nucleic acids encoding a number ofthe cytokines classified as “-interleukins”. These include, for example,interleukins 1 through 12. Also included in this category, although notnecessarily working according to the same mechanisms, are interferons,and in particular gamma interferon (γ-IFN), tumor necrosis factor (TNF)and granulocyte-macrophage-colony stimulating factor (GM-CSF). Otherpolypeptides include, for example, angiogenic factors andanti-angiogenic factors. It may be desirable to deliver such nucleicacids to bone marrow cells or macrophages to treat enzymaticdeficiencies or immune defects. Nucleic acids encoding growth factors,toxic peptides, ligands, receptors, or other physiologically importantproteins can also be introduced into specific target cells.

[0086] The recombinant retrovirus of the invention can be used for thetreatment of a neuronal disorder for example, may optionally contain anexogenous gene, for example, a gene which encodes a receptor or a genewhich encodes a ligand. Such receptors include receptors which respondto dopamine, GABA, adrenaline, noradrenaline, serotonin, glutamate,acetylcholine and other neuropeptides, as described above. Examples ofligands which may provide a therapeutic effect in a neuronal disorderinclude dopamine, adrenaline, noradrenaline, acetylcholine,gamma-aminobutyric acid and serotonin. The diffusion and uptake of arequired ligand after secretion by an infected donor cell would bebeneficial in a disorder where the subject's neural cell is defective inthe production of such a gene product. A cell genetically modified tosecrete a neurotrophic factor, such as nerve growth factor, (NGF), mightbe used to prevent degeneration of cholinergic neurons that mightotherwise die without treatment. Alternatively, cells be grafted into asubject with a disorder of the basal ganglia, such as Parkinson'sdisease, can be modified to contain an exogenous gene encoding L-DOPA,the precursor to dopamine. Parkinson's disease is characterized by aloss of dopamine neurons in the substantia-nigra of the midbrain, whichhave the basal ganglia as their major target organ.

[0087] Other neuronal disorders that can be treated similarly by themethod of the invention include Alzheimer's disease, Huntington'sdisease, neuronal damage due to stroke, and damage in the spinal cord.Alzheimer's disease is characterized by degeneration of the cholinergicneurons of the basal forebrain. The neurotransmitter for these neuronsis acetylcholine, which is necessary for their survival. Engraftment ofcholinergic cells infected with a recombinant retrovirus of theinvention containing an exogenous gene for a factor which would promotesurvival of these neurons can be accomplished by the method of theinvention, as described. Following a stroke, there is selective loss ofcells in the CA1 of the hippocampus as well as cortical cell loss whichmay underlie cognitive function and memory loss in these patients. Onceidentified, molecules responsible for CA1 cell death can be inhibited bythe methods of this invention. For example, antisense sequences, or agene encoding an antagonist can be transferred to a neuronal cell andimplanted into the hippocampal region of the brain.

[0088] For diseases due to deficiency of a protein product, genetransfer could introduce a normal gene into the affected tissues forreplacement therapy, as well as to create animal models for the diseaseusing antisense mutations. For example, it may be desirable to insert aFactor IX encoding nucleic acid into a retrovirus for infection of amuscle or liver cell.

[0089] The present invention also provides gene therapy for thetreatment of cell proliferative or immunologic disorders. Such therapywould achieve its therapeutic effect by introduction of an antisense ordominant negative encoding polynucleotide into cells having theproliferative disorder, wherein the polynucleotide binds to and preventstranslation or expression of a gene associated with a cell-proliferativedisorder. Delivery of heterologous nucleic acids useful in treating ormodulating a cell proliferative disorder (e.g., antisensepolynucleotides) can be achieved using a recombinant retroviral vectorof the present invention.

[0090] In addition, the present invention provides polynucleotidesequence encoding a recombinant retroviral vector of the presentinvention. The polynucleotide sequence can be incorporated into variousviral particles. For example, various viral vectors which can beutilized for gene therapy include adenovirus, herpes virus, vaccinia,or, preferably, an RNA virus such as a retrovirus. The retroviral vectorcan be a derivative of a murine, simian or human retrovirus. Examples ofretroviral vectors in which a foreign gene (e.g., a heterologouspolynucleotide sequence) can be inserted include, but are not limitedto: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). All of these vectors can transfer or incorporate a gene for aselectable marker so that transduced cells can be identified andgenerated. By inserting a heterologous sequence of interest into theviral vector, along with another gene which encodes the ligand for areceptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Targetingis accomplished by using an antibody or ligand to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome or attached to a viralenvelope to allow target specific delivery of the retroviral vectorcontaining the heterologous polynucleotide. In addition, the retroviralvector can be targeted to a cell by utilizing a cell- or tissue-specificregulatory element contained in the LTR of the retroviral genome.Preferably the cell- or tissue-specific regulatory element is in the U3region of the LTRs. In this way, after integration into a cell, theretroviral genome will only be expressed in cells where the cell- ortissue-specific promoter is active.

[0091] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral genome, byconventional calcium phosphate transfection. The resulting cells releasethe retroviral vector into the culture medium.

[0092] In another embodiment, the invention provides a method oftreating a subject having a cell proliferative disorder. The subject canbe any mammal, and is preferably a human. The subject is contacted witha recombinant replication competent retroviral vector of the presentinvention. The contacting can be in vivo or ex vivo. Methods ofadministering the retroviral vector of the invention are known in theart and include, for example, systemic administration, topicaladministration, intraperitoneal administration, intra-muscularadministration, as well as administration directly at the site of atumor or cell-proliferative disorder and other routes of administrationknown in the art.

[0093] Thus, the invention includes various pharmaceutical compositionsuseful for treating a cell proliferative disorder. The pharmaceuticalcompositions according to the invention are prepared by bringing aretroviral vector containing a heterologous polynucleotide sequenceuseful in treating or modulating a cell proliferative disorder accordingto the present invention into a form suitable for administration to asubject using carriers, excipients and additives or auxiliaries.Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975) and The National Formulary XIV., 14th ed. Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's The PharmacologicalBasis for Therapeutics (7th ed.).

[0094] For example, and not by way of limitation, a retroviral vectoruseful in treating a cell proliferative disorder will include a chimerictarget specific ENV protein directed to a cell type of interest (e.g.,one having a cell proliferative disorder), GAG, and POL proteins, acell-specific promoter sequence in the U3 region of the LTR of theretroviral genome associated with a growth regulatory gene (e.g.,probasin or HER2), and all cis-acting sequence necessary forreplication, packaging and integration of the retroviral genome into thetarget cell. The heterologous sequence can be, for example, an antisensemolecule or a suicide protein that results in the death of a cell wherethe retroviral genome is actively transcribed.

[0095] The following Examples are intended to illustrate, but not tolimit the invention. While such Examples are typical of those that mightbe used, other procedures known to those skilled in the art mayalternatively be utilized.

EXAMPLES Example 1

[0096] Construction of Replication Competent Retroviruses

[0097] There have been few reports in the literature regarding thestability of insertions in the context of replication-competent MoMLV,and all of these have used insertion positions within the 3′ longterminal repeat sequence (LTR). Most of these insertions were deletedwithin one or two serial passages of the virus. In this case the sizeand nature of the inserted sequences seemed to have little correlationwith the stability of the vector, as small inserts were often deletedjust as quickly as larger inserts. One important consideration may bethe positioning of the insertion; as the reverse transcription processentails duplication of the U3 region of the 3′ LTR (FIG. 1), this mayresult in decreased stability of non-essential sequences inserted intothis position.

[0098] An infectious Mo-MLV proviral clone was excised with NheI fromplasmid pZAP and ligated to the plasmid backbone of retroviral vectorgIZIN. The IRES of encephalomyocarditis virus was amplified by PCR fromplasmid pEMCF and appended at its 3′ end to a polylinker byoverlap-extension PCR. Plasmids gIZIN and pEMC-F were kindly provided byJ. J. Hwang, University of Southern California. All PCR reactions werecarried out with Pfu DNA polymerase (Stratagene). The IRES-polylinkerwas then introduced into the Mo-MLV clone at the 3′ terminus of the envgene by overlap extension PCR. The resulting plasmid was termed g1ZD.The GFP gene of plasmid pEGFP-N1 (Clontech) was amplified by PCR andinserted into the multiple cloning site of g1ZD, producing g1ZD-GFP. Thehygromycin phosphotransferase gene of plasmid pTK-hygro (Clontech) wassimilarly introduced into g1ZD to produce g1ZD-hygro. The prefix p isomitted in the designation of the viruses derived from these plasmids.

[0099] Insertion of a transgene into a less sensitive position, and infact linking expression of the inserted transgene to viral codingsequences, might enhance the stability of the vector. Accordingly, anIRES sequence was inserted just downstream from the envelope message butupstream from the 3′ LTR (FIG. 2). An IRES derived fromencephalomyocarditis virus (EMCV) and a multiple cloning site wereinserted just 3′ of the envelope gene in a replication-competent MoMLVprovirus clone, g1ZD (wild type MoMLV, see FIG. 1). The g1ZD strain ofMoMLV virus is ecotropic (i.e., encoated by an envelope withmurine-specific binding tropism). This particular insert position waschosen because 1) the packaging signal is known to extend past the ATGof the gag gene, thus positioning a transgene just upstream of the gaggene would greatly impair packaging efficiency, 2) the gag and polcoding sequences are initially translated as a single polypeptide whichis then cleaved, thus positioning a transgene between these codingsequences would greatly impair proteolytic processing, 3) the 3′ end ofthe pol gene actually overlaps with the 5′ end of the env gene, and thisoverlap region contains a splice acceptor for the env transcript, thustransgene insertions into this region would be problematic, and 4) thepositioning of the insert outside of the major intron ensures theinsert's presence on both spliced and unspliced viral RNAs and thereforethe translation of the insert from both spliced and unspliced RNAs.

[0100] The resultant construct was designated g1ZD. The multiple cloningsite in g1ZD was then used to insert transgene coding sequences. Themultiple cloning site in the gIZD was then used to insert transgenecoding sequences. Initially maker genes such as the green fluorescentprotein (GFP) gene, puromycine-resistance (puro^(R)) gene, andhygromycine-resistance (hygro^(R)) gene were inserted at this site.Suicide genes, such as the Herpes simplex virus thymidine kinase(HSV-tk) gene and the E. Coli purine nucleotide phosphorylase (PNP)gene, can be inserted in place of the marker genes. As the transgenesare of various sizes, resulting in IRES+transgene cassette insertionsraging from 1170 bp to 1700 bp in size, it can be determined whether theinset size has an effect on the stability of the virus genome (normally8.3 kb in size), and what the packaging limit for MoMLV might be in thiscontext. There have been few reports in the literature regarding thestability of insertions in the context of replication-competent MoMLV,and none, that the inventors are aware of, using an IRES sequence todirect transgene expression in replication-competent vectors. Thisconstruct design greatly improves functional and genetic stability ofthe transgene.

[0101] The gIZD-derived replication-competent retrovirus (RCR) vectorswere first tested for their ability to efficiently replicate and spreadin culture. NIH3T3 and 293T cell were cultivated in Dulbecco's ModifiedEagle Medium with 10% fetal bovine serum. Vector stock was produced bytransfection of the vector-encoding plasmids into 293T cells usingcalcium phosphate-precipitation as described previously. Twenty-fourhours post-transfection, the medium was replaced with fresh medium, andone day later the vector-containing supernatant was collected, filteredthrough a 0.45 μm filter and used immediately or frozen for later use.After initial transfection of the RCR vector plasmids into 293 cells toproduce a viral stock, a 1000-fold dilution of the virus preparation wasused to infect fresh plates of NIH3T3 cells. The cells were grown toconfluence, the RCR-containing cell culture supernatant was harvested toassay reverse transcriptase (RT) activity, and the cells were thenpassaged. This cycle was repeated several times as each set of passagedcells again attained confluence.

[0102] Dilutions of vector stocks were used to infect 20% confluentNIH3T3 cells. Every 3 days for the following 2 weeks, the supernatantwas collected and the cells were split 1:4. To quantitate reversetranscriptase activity, an aliquot of each supernatant was incubated at37° C. for one hour in a cocktail containing (³²P)dTTP, poly(rA)template, and oligo-dT primers. RT activity was quantified usingpoly-riboA template and an oligo-dT primer for incorporation ofradiolabeled dTTP, and the reaction products were spotted onnitrocellulose and radioactivity measured by PhosphoImager. The timecourse of RT activities over several passages shows a classic peak andplateau pattern, thus indicating that all gIZD-derived RCR vectorscarrying marker genes are capable of efficient replication and spreadthroughout a cell culture at levels comparable to wild-type virus (FIG.3). Thus, even relatively large insertions, that stretch the packagingcapacity of MoMLV to its limit, do not appear to impair the replicativeability of the virus.

[0103] The gIZD-derived RCR vector containing GFP as the marker gene(gIZD-GFP) was used to follow transgene expression over time as thevirus spread through the NIH3T3 cell culture. The GFP marker can bedetected by fluorescence-activated cell sorter (FACS) analysis, usingthe same wavelength as that used for detection of fluorescein (FITC;cannel FL1). NIH3T3 cells were washed with phosphate buffered saline(PBS), trypsinized and collected by low-speed centrifugation. Cells wereresuspended in PBS at approximately 10⁵ cells/ml and analyzed forfluorescence with a Becton Dickinson FACScan using a fluoresceinisothiocyanate filter set. These results show that initial transductionlevels at high dilution of the virus stock are extremely low (about 3%)at Day 3, but expression in the culture rapidly increased over time, asseen by the shifted peak of mean fluorescence, so that by Day 8 almost100% of the culture is now expressing GFP (FIG. 4). Thus, this indicatesthat transgene expression is not lost as the RCR vector spreads throughthe cell culture, and in fact the transgene is efficiently delivered topractically all of the cells even with low initial transduction levels(FIGS. 5 and 6).

Example 2

[0104] Construction of RCR Vectors Targeted to Human Breast Cancer Cells

[0105] Chimeric MoMLV and SNV env sequences which contain targetingmoieties directed against human breast cancer cells, and which haveproven successful for targeting in previous studies, were utilized. Thetargeting moiety for MoMLV env was the peptide hormone heregulin, andthe targeting moiety for SNV env was the single chain antibody B6.2,originally derived by immunizing mice with a membrane-enriched fractionfrom a human breast tumor. Using an IRES sequence, either a marker gene(such as GFP) or a suicide gene (such as HSV-tk or PNP) is linked to thechimeric envelope construct at the 3′ end. These chimericenvelope/IRES/transgene constructs are then recloned back into thereplication-competent wild type forms of MoMLV or SNV, replacing theoriginal env gene (FIG. 2b). This results in replication-competentretrovirus vectors that are targeted specifically to cancer cells.

[0106] Tumors were established by the injection of 1.5×10⁶ NMU ratbreast adenocarcinoma cells subcutaneously into the anterior flanks of6-week-old nu/nu BALB/c mice (Simonsen Laboratories). Four weeks later,the tumors had grown to 100-150 mm³, at which time they were injectedwith 80 μl of supernatant from glZD-GFP infected NIH3T3 cells,containing 1×10⁴ PFU vector. At regular intervals thereafter, subsets ofthe mice were sacrificed, and their tumors were surgically removed. Toproduce single-cell suspensions, the entire mass of each tumor wasfinely minced and incubated for one hour at 37° C. in five volumes ofHank's balanced salt solution (HBSS) containing 100 U/ml collagenase IV.The dispersed cells were then washed and resuspended in PBS for flowcytometric-analysis.

[0107] Although Kasahara et al. (Science, 266:1373-1376 (1994)) and Chuet al. (Journal of Virology, 69:2659-2663 (1995)) have found that theco-expression of wild-type MoMLV envelope is usually required for properprocessing and transport of chimeric MoMLV envelope constructs to thesurface of the producer cells, and possible also for proper functionduring entry, some groups have been able to encoat virions with chimericenvelope alone by inserting the ligand sequence into the extremeamino-terminus of the MoMLV envelope. The strategy was used forconstruction of the heregulin/MoMLV envelope to be used in thereplication-competent vectors. Furthermore, although MoMLV is thestandard retrovirus used in most gene therapy protocols, SNV isadvantageous for targeting due to the following characteristics: 1) itsmaximum packaging capacity is larger than that of MoMLV and may thustolerate the additional sequences and genes without drastic loss oftiter, 2) the SNV envelope has been found to be extremely stable,tolerating major truncations without loss of the ability to assembleproperly on the packaging cell surface and it has been shown thechimeric SNV en lope constructs contain exogenous ligand sequences canbe expressed without the need for wild type SNV envelope, and 3) wildtype SNV is considered to be completely non-pathogenic for humans (Bacuset al., Am. J. Clin. Pathol., 102:S13-24 (1994)).

Example 3

[0108] Creation of RCR Vector-producing Cell Lines

[0109] The above MoMLV and SNV genomic constructs are transfected intohuman breast cancer cell line MD-MB-453 (ATCC accession umber HTB 131),which expresses high levels of HER-2 and HER-4 (Krause et al., EMBOJournal, 6:605-610 (1987)). The constructs which contain the GFP markergene are transfected first, as the presence of the marker gene enablesus to monitor the transfection efficiency by FACS analysis. Aftertransfection, the targeted RCR vectors produced by the primarytransfectants are capable of horizontal infection of adjacent cells notinitially transfected, by biding via the heregulin or B6.2 single-chainantibody moieties. This can be detected as an increasing percentage ofGFP-positive cells over time. Furthermore, the cell culture mediumshould contain supernatant virus, which can infect and transduce freshcultures of MDA-MB-453 cells. The GFP-containing vectors thus enable oneto determine the time course of transfection and infection events, andrate of virus spread through the human breast cancer cell culture. Basedon this information, similar studies are performed with the HSV-tk- andPNP-containing vectors, and in this case transduction is monitored bySouthern blot or quantitative PCR for integrated vector sequences. TheHSV-tk and PNP transgenes are also functionally tested by determiningwhether sensitivity had been conferred to the prodrugs ganciclovir and6-methyl purine-deoxyriboside, respectively.

Example 4

[0110] Testing of Tissue Specificity of the Virus in Culture

[0111] Cell culture medium from virus-producing MDA-MB-453 cells is usedto infect a variety of human target cells, in order to ascertain thetissue-specificity of the virus vectors. As negative control virus, thetarget cells are exposed to wild type ecotropic MoMLV or SNV vectorscontaining the GFP marker gene, and as positive control virus, thetarget cells are infected with an amphotropic MoMLV vector containingGFP.

[0112] The target cells again are the human breast cancer cell lineMDA-MB-453, which as noted above over expresses both HER-2 and HER-4,and as a negative control cell line, the human breast cancer cell lineMDA-MB-231 (ATCC accession number HTB 26), which does not express anydetectable HER-2 or HER-4 is used. No background infectivity is seenwith the wild type ecotropic MoMLV or SNV vector controls; thussuccessful infection by the chimeric vectors depends on specificinteraction between the heregulin or B6.2 single-chain antibodytargeting moieties in the virus envelope and their correspondingreceptor or antigen on the target cells. Other human breast cancer celllines that are used as targets include BT474 (which over expresses bothHER-2 and HER-4) and MCF7 (which only expresses HER-4). In addition,negative control cell lines which are of human origin but not derivedfrom mammary epithelium are used to further test tissue-specificity ofinvention.

[0113] As noted above, infected cells are examiner by FACS analysis forGFP expression, or tested for transduction of HSV-tk or PNP by Southernblot or quantitative PCR and by exposure to ganciclovir or 6-methylpurine-deoxyriboside.

Example 5

[0114] Targeting of RCRV's by Incorporation of Tissue-specific PromoterElements

[0115] Retroviral tropism can be re-directed by altering thetranscriptional activity of the virus through replacement of regions ofthe viral long terminal repeat (LTR) with cell-specific promoterelements. This strategy has been used by other groups to targetretroviral transcription to particular tumor cell types.

[0116] The MoMLV proviral LTR sequences consist of 3 distinct regions,designated U3, R, and U4, which are repeated at each end of the genome.The promoter elements that control transcription of the RNA genome andtherefore replication of the virus, reside in the U3 region. The Rregion contains the start site of transcription, and therefore theupstream U3 region is not included in the genomic RNA transcript.However, the transcript reads through to the U3 sequence into the 3′LTR, which also contains polyadenylation signals, and the 3′ LTR U3region is re-duplicated at the 5′ end during the process of reversetranscription. Thus, for alterations in the LTR promoter to be permanentover serial cycles of replication, the alterations is incorporated intothe U3 region of the 3′ LTR.

[0117] In the present invention tissue specific elements areincorporated in the LTR 3′ U3 region in order to target RCR vectorreplication. As a practical example, transcription targeting to prostatecancer cells is shown. Using the specificity of trans-activatingprostate-specific elements which interact with cis-acting promotersequences (androgen response elements) investigators have been able toachieve tissue-specific transgenic targeting of oncogenic proteins(Greenberg et al., Proc Natl. Acad. Sci. USA, 92:3439-43 (1995); andGarabedian et al., Proc. Natl. Acad. Sci USA, 95:15382-7 (1998)). One ofthe most well-characterized proteins uniquely produced by the prostateand regulated by promoter sequences responding to prostate-specificsignals, is the rat probasin protein. Study of the probasin promoterregion has identified tissue-specific transcriptional regulation sites,and has yielded a useful promoter sequence for tissue-specific geneexpression. The probasin promoter sequence containing bases −426 to +28of the 5′ untranslated region, has been extensively studied in CATreporter gene assays (Rennie et al., Mol Endo, 7:23-36 (1993)).Prostate-specific expression in transgenic mouse models using theprobasin promoter has been reported (Greenberg et al., Mol Endo, 8:230-9(1994)). Gene expression levels in these models parallel the sexualmaturation of the animals with 70 fold increased gene expression foundat the time of puberty (2-6 weeks). Castration of the animals will dropgene expression to near zero which can be increased to pre-castratelevels following the parenteral administration of testosterone. Theprobasin promoter (−426 to +28) has been used to establish the prostatecancer transgenic mouse model that uses the fused probasinpromoter-simian virus 40 large T antigen gene for targeted overexpression in the prostate of stable transgenic lines (Greenberg et al.,Proc Natl. Acad. Sci. USA, 92:3439-43 (1995)). Thus, this region of theprobasin promoter is incorporated into the 3′ LTR U3 region of the RCRvectors. Thus providing a replication-competent MoMLV vector targeted bytissue-specific promoter elements.

Example 6

[0118] Incorporation of Prostate-specific Promoter Elements in to theRCRV LTR

[0119] A fragment of the rat probasin androgen-sensitive promoter (from−426 to +28) that has been shown to specify prostate-specific geneexpression has been engineered into the U3 region of the retroviral 3′LTR in both ecotropic and amphotropic RCR vectors. The 5′ end of the U3region is recognized by viral integrase protein and so overlap extensionPCR was used to precisely place the probasin promoter just downstream ofthe beginning of the U3 region in the 3′ LTR, replacing the rest of theU3 sequence up to the R region. Since it is initially placed downstream,this modified U3 region will not be operative upon transfection of theprovirus construct into 293 cells and production of the vectortranscript will proceed normally, but after a single round ofreplication the probasin sequence will be re-duplicated in the U3 regionof the 5′ LTR (FIG. 8), and thereafter should specify prostatecell-specific replication of the virus. Probasin-targeted RCR vectorshave been constructed containing the EMCV IRES-GFP marker gene cassette;in this case the U3 region in the 5′ LTR of g1ZD-GFP was first replacedwith a CMV promoter (c1ZD-GFP) to remove the Nhe I site in the 5′ LTR(and also to enhance expression and titers after initial transfection in293 cells), so that the Nhe I site in the 3′ LTR is now unique, and canbe used to insert the probasin promoter fragment (FIG. 9A: replacementof the 3′ U3 region with the probasin promoter by overlap extension PCR;FIG. 9B: sequence of the 3′ LTR in p1ZD-GFP, showing the probasinpromoter/R region joint). It should be noted in this context that,although insertions of non-essential transgenes in the U3 region areindeed prone to deletion, the probasin promoter in this case willcompletely replace the wild type promoter elements in the viral LTR,therefore deletions of the probasin promoter would simply result in avirus that is unable to replicate, thus there would be selectionpressure against such deletions. To the inventors' knowledge this wouldrepresent the first example of a replicating retroviral vectorcontrolled by transcriptional regulation.

[0120] A fragment of the rat probasin androgen-sensitive promoter wasconstructed by polymerase chain reaction (PCR) amplification fromgenomic DNA using primers ATCCACAGTTCAGGTTCAATGGCG andCTGCTACCTTCTTTTTGA GATTCTTGTCTGTCATCATACTGG. As discussed above, this isthe same promoter fragment (from −426 to +28) that specifiesprostate-specific oncogene expression in the probasin-SV40 T antigentransgenic mouse. A NheI-SfiI linker sequence was added to the 5′ primerwhile an AflII site was added to the 3′ end of the 3′ primer. This PCRproduct was inserted into the pcDNA3.+expression plasmid (Invitrogen)following a NheI-AflII digestion. The presence of the probasin insertwas confirmed by restriction digest with NheI-AflII to isolated the 550bp fragment.

[0121] This probasin promoter sequence is engineered into the U3 regionof the retroviral 3′ LTR by overlap extension PCR in both gIZD-GFP andgIZA-GFP and also in the GIZD and gIZA vector constructs that containthe PNP or HSV-tk therapeutic genes. The 5′ end of the U3 region isrecognized by the viral integrase protein and,so overlap extension PCRwill be used to precisely place the probasin promoter just downstream ofthe beginning of the U3 region in the 3′ LTR, replacing the rest of theU3 sequence up to the R region. This modified U3 will not be operativeupon initial transection of the RCR vector construct into 293 cells, butafter one round of replication the probasin sequence will bere-duplicated in the U3 region of the 5′ LTR, and thereafter shouldspecify prostate cell-specific replication of the virus. The constructis transfected into 293 cells, and the supernatant harvested to test thecell type-specificity of viral replication, as described below.

Example 7

[0122] Testing the Tissue Specificity of the Transcriptionally TargetedRCRV in Culture:

[0123] In order to confirm that the 430-bp probasin promoter would stillbe capable of prostate-specific, androgen-inducible expression afterbeing incorporated into the retroviral long terminal repeat (LTR), thishybrid promoter was constructed and used to drive expression of aluciferase reporter gene. As shown in FIG. 10, this construct was testedin both prostatic and non-prostatic cell lines, in the presence andabsence of androgen stimulation. A representative set of results isshown in FIG. 11; the results confirm that the probasin-LTR hybridpromoter is active with androgen stimulation only in prostate celllines, whereas non-prostatic cell lines show little activity even in thepresence of androgen stimulation. Similar results were obtained with theother cell lines tested.

[0124] The probasin-LTR hybrid promoter was then incorporated into RCRvectors carrying the green fluorescent protein (GFP) marker gene, byreplacement of the 3′ LTR so that probasin-driven expression would occuronly after one round of reverse transcription and re-duplication of the3′ LTR at the 5′ end. As shown in FIG. 12, virus preparations weregenerated from these constructs by harvesting the supernatant medium48-72 hours after transient transfection of 293T cells. These viruspreparations were filtered to exclude cell debris, and then used toinfect murine prostate cancer (TRAMP-C) cell lines. GFP expression inthe infected TRAMP-C cells was examined by fluorescence-activated cellsorter (FACS) analysis in the presence and absence of androgenstimulation. As shown in FIG. 13, a shift in fluorescence indicatingexpression of the GFP marker gene occurred in the infected prostatecells only upon androgen stimulation.

[0125] In addition, the genomic constructs were used for infection ofthe human prostate cancer cell line LnCaP, which expresses high levelsof the prostate-specific membrane antigen (PSMA) and supports high levelexpression of the probasin promoter. The RCR vectors which contain theGFP marker gene were used for infection first, as the presence of themarker gene will enable one to monitor the transduction efficiency byFACS analysis. After initial transduction, the targeted RCR vectorsproduced by the primary transfectants are capable of horizontalinfection of adjacent cells not initially transfected. This is detectedas an increasing percentage of GFP-positive cells over time.Furthermore, the cell culture medium will contain supernatant virus,which can infect and transduce fresh cultures of LnCaP cells. TheGFP-containing vectors thus enabled determination of the time course oftransfection and infection events and rate of virus spread through theprostate cancer cell culture. Based on this information, similar studiesare performed with the HSV-tk- and PNP-containing suicide gene vectors,and in this case transduction is monitored by Southern blot orquantitative PCR for integrated vector sequences. The HSV-tk and PNPtransgenes are also functionally tested by determining whethersensitivity is conferred to the prodrugs ganciclovir and6-methylpurine-deoxyriboside, respectively.

[0126] Targeted RCR vectors are also used to infect a variety ofnon-prostatic target cells, in order to confirm the tissue-specificityof the virus vectors. As a control virus, the target cells are exposedto wild-type ecotropic or amphotropic MoMLV vectors containing the GFPmarker gene.

Example 8

[0127] Transduction of Prostate Tumors in vivo:

[0128] To study in vivo transduction a number of models are availablethat mimic the various clinical aspects of prostate cancer and includespontaneous rodent models, human xenograft systems usingimmunocompromised murine hosts and murine transgenic models. The DunningR-3327 rodent model for adenocarcinoma of the prostate involves the useof subcutaneously implanted tumors in Copenhagen rats (Dunning, W.,Natl. Cancer Inst., 12:p351 (1963)). This model allows the study ofandrogen independent progression and the process of metastasis formationusing the MAT-Lylu or MAT-lu sublines (Smolev et al., Cancer Treat.Rep., 61, 273 (1977)).

[0129] In addition, the successful development of transgenic animalmodels that are capable of the spontaneous development of prostatecancers that resemble human adenocarcinomas have relied on thetissue-specific transgene expression. In particular, the probasinpromoter driving the SV40 T-antigen has been used to establish aprostate cancer transgenic mouse model. This well worked out modeldemonstrates spontaneous prostate tumors histologically similar to thosethat develop in humans, although it lacks the underlying hormonal basisthought o play a central role in prostate tumor initiations. The in vivoefficacy of the transcriptionally targeted RCRV's can be shown usingthis model.

[0130] Male transgenic mice at puberty are monitored of the developmentof prostate tumors. The tumors are injected with the targetedreplication-competent MoMLV or SNV vectors carrying GFP or with negativeand positive control virus preparations, and transduction assessed afteranother two weeks. At that time, the animals are sacrificed and thetumors harvested. Tissue samples from tumors exposed to viral vectorscarrying the GFP gene are snap frozen in liquid nitrogen and frozensections examined histologically under UV fluorescence microscopy. Basedon these results, similar experiments are performed using ht targetedRCR vectors carrying HSV-tk or PNP. In this case, tow weeks after thexenografts are exposed to viral vectors carrying the HSV-tk or PNP gene,ganciclovir or 6-methyl purine-deoxyriboside is administered to theanimals and the extent of shrinkage of the tumors assessed. Controlgroups are left untreated as a control for tumor growth.

[0131] Increased transduction efficiency by the use oftarget-restricted, replication-competent retroviral vectors wouldrepresent a significant improvement in vector design. As the initiallyinfected tumors cells in turn produce more virus, this strategy takesadvantage of the amplification process inherent in the wild-type viruslife cycle. Targeting the retrovirus specifically and exclusively totumors cells limits and controls the replicative process, and the use ofnormally non-pathogenic viruses as the basis for these vectors, as wellas the incorporation of suicide gene in the vectors as a “self-destruct”mechanism, provide further safeguards which minimize the risk to normalcells.

Example 9

[0132] Intra-tumoral Spread of the RCRV's in Breast Cancer Model

[0133] The in vivo application of replication-competent MoMLV vectors byintra-tumoral injection into solid tumors derived from rat NMU cells(nitrosomethylurea-induced breast cancer) in a nude mouse subcutaneousxenograft model was also performed. NMU cells are known to betumorigenic in nude mice. Nude mice were anesthetized and a subcutaneousinjection of 2×10⁶ NMU cells in PBS suspension was performed toestablish tumors. The tumors were allowed to grow to approximately 1 cmin diameter over a period of 4 weeks, at which point the g1ZD-GFP RCRvector was administered by intra-tumoral injection of 100 μl of thevector preparation.

[0134] The titer of the g1ZD-GFP vector preparation was 10⁵/ml titer byXC cell syncytia assay, therefore this constitutes a total inoculum ofonly 10⁴ infectious units of virus. In this instance, taking intoaccount the tumor growth and cell division following initialestablishment, a conservative estimate of the multiplicity of infection(MOI) would be on the order of at least 0.001 (and perhaps more likelyto be on the order of 0.0001). Thus the initial transduction efficiencywould be expected to be as low as, or lower than, 0.1%. Again, thisinitial inoculum of virus supernatant is comparable to the lowtransduction efficiencies obtained in the clinical trials usingintra-tumoral injection of PA317 packaging cell lines to transduceglioblastoma.

[0135] Tumors were allowed to grow for various intervals after vectorinjection, and a set of mice was sacrificed and the tumors wereharvested at 2 week, 4 week, and 6 week time points post-injection.After tumor harvest, the tumors were sectioned and some tumor sampleswere immediately frozen for subsequent isolation of genomic DNA andSouthern blot analysis. The other tumor samples were minced, immediatelytreated with collagenase for 3-4 hours to disaggregate the tumor cellswhile still viable, washed and resuspended in PBS, and examined by FACSanalysis the same afternoon. Thus horizontal spread of the virus vectorafter disaggregation of the tumor cells is unlikely to have affected theresults, as there was not enough time elapsed for retroviral entry,integration, and GFP transgene expression to have occurred prior to FACSanalysis.

[0136] The results are shown in FIG. 7: although at the 2 week timepoint, only a small percentage of cells initially appear to show a shiftin fluorescence, highly efficient gene transfer throughout the entiretumor is evidenced by FACS analysis of disaggregated tumor cells 4-6weeks after injection of the initial inoculum. Intact, full-lengthgenomic bands were detected in Southern blots of proviral DNA isolatedfrom individual tumors at the 4 and 6 week time points. This indicatesthat the RCR vector was capable of efficient replication and genedelivery in the context of solid tumors in vivo without deletionsoccurring during this time interval, and provides an illustrativeexample of the potential power of this strategy for cancer gene therapy,especially considering the extremely low MOI of the initial inoculum.

Example 10

[0137] IRES Sequence Variations

[0138] As described above, vectors were constructed varying the size ofthe viral genome. By using IRES sequences shorter than the EMCV IRESpresent in the constructs above, it may be possible to insert transgeneslarger than GFP or large cell type-specific targeting sequences. Two newvectors were constructed using IRES sequences from the BiP (Yang andSarnow, 1997) and VEGF (Stein et al., 1998) genes (FIG. 14). The BiPIRES-containing vector, ZB-GFP, and the VEGF IRES-containing vector,ZV-GFP, are 450 bp and 380 bp shorter than g1ZD-GFP, respectively.Infection of NIH3T3 cells by the vectors demonstrated that bothefficiently transduce cells and express GFP, although transgeneexpression levels are somewhat lower than with g1ZD-GFP (FIG. 15). Theability of ZB-GFP and ZV-GFP to retain their IRES-GFP sequences throughvector spread was determined by conducting serial infections of NIH3T3cells with the vectors. Proviral (Hirt) DNA was prepared from 13serially infected NIH3T3 populations and was subjected to Southernanalysis using a probe for the LTR-gag region of Mo-MLV. FIG. 16 showsthat the IRES-GFP sequences of both of the new vectors were retained forapproximately the same number of serial infections as that of ZAPd-GFP.This indicated that a reduction in the size of the IRES in these vectorsdoes not significantly alter vector stability, but may allow theinsertion of transgenes larger than GFP.

Example 11

[0139] RCR Vectors Targeted to Breast Tumor Cells Using Two Types ofModification to the Envelope Protein

[0140] In order to obtain RCR vectors targeted to breast tumor cells,vectors were constructed containing modifications in the envelope genethat would allow specific binding of vector particles to proteinsexpressed on the surface of breast tumor cells. Two approaches intargeting the vectors were used. The first approach involves insertionof sequences encoding the IgG-binding domain (“Z domain,” Nilsson etal., 1987) of the S. aureus protein A into the proline-rich region (PRR,or “hinge”) of the envelope gene (FIG. 17). The presence of the Z domainon the vector surface would allow the binding of tumor-specificantibodies to the vector and would therefore presumably allow specificbinding of the vector to tumor cells via the antibody. The secondapproach involves the replacement of the wild type receptor bindingdomain (RBD) of the envelope with sequences encoding a single-chainantibody (scFv) against HER2 (kindly provided by Drs. Michael Press andJinha Park), (FIG. 18). This modification is expected to ablate bindingof the vector to its normal receptor while allowing direct binding toHER2-expressing tumor cells.

[0141] Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1 8 1 24 DNA Mouse 1 atccacagtt caggttcaat ggcg 24 2 18 DNA ArtificialSequence synthetic oligonucleotide 2 ctgctacctt ctttttga 18 3 24 DNAMouse 3 gattcttgtc tgtcatcata ctgg 24 4 31 DNA homo sapien 4 agagtacgagccatagataa cgttactggc c 31 5 31 DNA homo sapien 5 cacgataata ccatggccattcgaacaaag g 31 6 31 DNA Homo sapien 6 ctgtacaagt agcggccgcg ccatagataaa 31 7 31 DNA Homo sapien 7 cacgataata ccatggccat tcgaaccgcg a 31 8 31DNA Homo sapien 8 tgggggccgc gccatagata aaataaaaga t 31

What is claimed is:
 1. A recombinant replication competent retrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral ENV protein; a retroviral genome comprising Long-Terminal Repeat (LTR) sequences at the 5′ and 3′ end of the retroviral genome, wherein a target specific polynucleotide sequence is contained within the LTR sequences at the 5′ and/or 3′ end of the retroviral genome, a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and cis-acting nucleic acid sequences necessary for reverse transcription, packaging and integration in a target cell.
 2. The retrovirus of claim 1, wherein the retroviral genome is derived from a lentivirus.
 3. The retrovirus of claim 2, wherein the lentivirus is human immunodeficiency virus (HIV).
 4. The retrovirus of claim 1, wherein the retroviral genome is derived from the group consisting of murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Gibbon ape leukemia virus (GALV) and Human Foamy Virus (HFV).
 5. The retrovirus of claim 4, wherein the MLV is an amphotropic MLV.
 6. The retrovirus of claim 1, wherein the ENV protein comprises an ENV sequence present in the group consisting of murine leukemia virus (MLV) and Vesicular stomatitis virus (VSV) ENV.
 7. The retrovirus of claim 1, wherein the ENV protein further comprises a target-specific ligand sequence.
 8. The retrovirus of claim 7, wherein the targeting specific ligand sequence is an antibody, receptor, or ligand.
 9. The retrovirus of claim 6, wherein the ENV sequence is an amphotropic protein.
 10. The retrovirus of claim 6, wherein the ENV sequence is a ecotropic protein.
 11. The retrovirus of claim 1, wherein the target cell is a cell having a cell proliferative disorder.
 12. The retrovirus of claim 1, wherein the target cell is a neoplastic cell.
 13. The retrovirus of claim 11, wherein the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer and ovarian cancer.
 14. The retrovirus of claim 1, wherein the target specific polynucleotide sequence is a tissue-specific promoter sequence.
 15. The retrovirus of claim 14, wherein the promoter sequence is associated with a growth regulatory gene.
 16. The retrovirus of claim 1, wherein the heterologous polynucleotide sequence is a suicide gene.
 17. The retrovirus of claim 15, wherein the suicide gene is a thymidine kinase.
 18. The retrovirus of claim 1, wherein the heterologous sequence is a marker gene.
 19. The retrovirus of claim 1, wherein the regulatory nucleic acid sequence operably associated with the heterologous nucleic acid sequence is selected from the group consisting of a promoter, an enhancer, and an internal ribosome entry site.
 20. A recombinant retroviral polynucleotide, comprising: a polynucleotide sequence encoding a GAG protein; a polynucleotide sequence encoding a POL protein; a polynucleotide sequence encoding an ENV protein; a polynucleotide sequence comprising a Long Terminal Repeat (LTR) at the 5′ and 3′ end of the retroviral polynucleotide sequence containing a target specific polynucleotide sequence; a heterologous polynucleotides sequence operably linked to a regulatory nucleic acid sequence; and cis acting polynucleotide sequence necessary for reverse transcription, packaging and integration in a target cell.
 21. The polynucleotide of claim 20, wherein the GAG, POL and ENV sequences are derived from a lentivirus.
 22. The polynucleotide of claim 21, wherein the lentivirus is human immunodeficiency virus (HIV).
 23. The polynucleotide of claim 20, wherein the GAG, POL and ENV polynucleotide sequences are derived from murine leukemia virus (MLV) or Moloney murine leukemia virus (MoMLV).
 24. The polynucleotide of claim 23, wherein the MoMLV is an amphotropic MoMLV.
 25. The polynucleotide of claim 20, wherein the ENV sequence is derived from the group consisting of murine leukemia virus (MoMLV) and Vesicular stomatitis virus (VSV) ENV.
 26. The polynucleotide of claim 20, wherein the ENV sequence further comprises a target-specific ligand polynucleotide sequence.
 27. The polynucleotide of claim 26, wherein the targeting specific ligand sequence encodes an antibody, receptor, or ligand.
 28. The polynucleotide of claim 25, wherein the ENV sequence is an amphotropic protein.
 29. The polynucleotide of claim 25, wherein the ENV sequence is an ecotropic protein.
 30. The polynucleotide of claim 20, wherein the target cell has a cell proliferative disorder.
 31. The polynucleotide of claim 20, wherein the target cell is a neoplastic cell.
 32. The polynucleotide of claim 30, wherein the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer, thyroid cancer, liver cancer, and brain cancer and ovarian cancer.
 33. The polynucleotide of claim 20, wherein the target specific polynucleotide sequence is a cell- or tissue-specific promoter sequence.
 34. The polynucleotide of claim 33, wherein the promoter sequence is associated with a growth regulatory gene.
 35. The polynucleotide of claim 20, wherein the heterologous polynucleotide sequence is a suicide gene.
 36. The polynucleotide of claim 35, wherein the suicide gene is a thymidine kinase or a purine nucleoside phosphorylase (PNP).
 37. The polynucleotide of claim 20, wherein the heterologous sequence is a marker gene.
 38. The polynucleotide of claim 20, wherein the regulatory nucleic acid sequence operably associated with the heterologous nucleic acid sequence is selected from the group consisting of a promoter, an enhancer, and an internal ribosome entry site.
 39. The polynucleotide of claim 20, wherein the polynucleotide sequence is contained in a viral particle.
 40. The polynucleotide of claim 20, wherein the polynucleotide sequence is contained in a pharmaceutically acceptable carrier.
 41. A method of treating a subject having a cell proliferative disorder, comprising: contacting the subject with a retrovirus, comprising, a retroviral GAG protein; a retroviral POL protein; a retroviral ENV protein; a retroviral genome comprising Long-Terminal Repeat (LTR) sequences at the 5′ and 3′ end of the retroviral genome, wherein a target specific polynucleotide sequence is contained within the LTR sequences at the 5′ and 3′ end of the retroviral genome, a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and cis-acting nucleic acid sequences necessary for reverse transcription, packaging and integration in a target cell.
 42. The method of claim 41, wherein the subject is a mammal.
 43. The method of claim 42, wherein the mammal is a human.
 44. The method of claim 41, wherein the contacting is by in vivo administration of the retrovirus.
 45. The method of claim 44, wherein the in vivo administration is by systemic, local, or topical administration.
 46. The method of claim 41, wherein the contacting is by ex vivo administration of the retrovirus.
 47. The method of claim 41, wherein the retroviral genome is derived from a lentivirus.
 48. The method of claim 47, wherein the lentivirus is human immunodeficiency virus (HIV).
 49. The method of claim 41, wherein the retroviral genome is derived from murine leukemia virus (MLV) or Moloney murine leukemia virus (MoMLV).
 50. The method of claim 49, wherein the MoMLV is an amphotropic MoMLV.
 51. The method of claim 41, wherein the ENV protein contains an ENV sequence selected from the group consisting of Moloney leukemia virus (MoMLV) and Vesicular stomatitis virus (VSV) ENV.
 52. The method of claim 41, wherein the ENV protein further comprises a target-specific ligand sequence.
 53. The method of claim 52, wherein the targeting specific ligand sequence is an antibody, receptor, or ligand.
 54. The method of claim 41, wherein the target cell is a cell having a cell proliferative disorder.
 55. The method of claim 41, wherein the target cell is a neoplastic cell.
 56. The method of claim 54, wherein the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer and ovarian cancer.
 57. The method of claim 1, wherein the target specific polynucleotide sequence is a tissue-specific promoter sequence.
 58. The method of claim 41, wherein the promoter sequence is associated with a growth regulatory gene.
 59. The method of claim 41, wherein the promoter sequence is associated with probasin.
 60. The retrovirus of claim 41, wherein the heterologous polynucleotide sequence is a suicide gene.
 61. The retrovirus of claim 41, wherein the suicide gene is a thymidine kinase.
 62. A recombinant replication competent murine leukemia virus (MLV), comprising: an MLV GAG protein; an MLV POL protein; an MLV ENV protein; an MLV genome comprising Long-Terminal Repeat (LTR) sequences at the 5′ and 3′ end of the retroviral genome, a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and cis-acting nucleic acid sequences necessary for reverse transcription, packaging and integration in a target cell. 